This invention relates to peptide derivatives and processes for measuring activities of physiologically active substances by using the same as substrates
Substances composed mainly of amino acids such as peptide hormones, enzymes, and the like fulfill various important physiological functions in living bodies. When these physiological functions appear, it is known that there take place in living bodies specific modifications of peptides such as, cleavages of peptide bonds, addition of sugars, phosphoric acid groups, sulfonic acid groups, sulfuric acid groups, etc. to peptides, and also specific elimination reactions of some groups. For example, there are concretely known activation of polypeptide hormones by limited proteolysis, controls of biological activities of proteins by phosphorylation, cleavage of the leader peptide, addition or elimination of sugar chains to proteins, etc. Elucidation of these modifications is medically and physiologically very important. Various studies have therefore been carried out as to physiologically active substances causing such reactions.
In activity measurements of physiologically active substances acting on peptides, proteins, etc., it is most in general to measure the changes of the substrates by various actions due to the physiologically active substances such as modification, elimination, and decomposition using peptides or proteins as substrates. For example, in the case of measuring protease or peptidase activities, synthesized substrates introduced a chromogenic group into peptides or amino acids are used. As the chromogenic group, there can be used coumarin, naphthylamine, nitroaniline, hippuric acid, and derivatives of these compounds. The activity can be determined by measuring fluorescence intensity or a change of absorbance (absorption curve) of the chromogenic group liberated by hydrolysis of the bond between the chromogenic group and the amino acid, or by measuring absorbance of a coloring matter obtained by a color producing reaction of the liberated chromogenic group and a coupler. As mentioned above, the method comprising hydrolyzing the bond directly by the physiologically active substance and measuring the liberated chromogenic group is simple and easy. But since the portion which is hydrolyzed is the bond between the amino acid and the chromogenic group, such a method is not suitable for measuring a physiologically active substance high in specificity. Further, the application of this method is limited in number since the method can not applied for the physiologically active substances which do not accompany with peptide cleavage. Moreover, according to a process wherein a peptide portion of a substrate is hydrolyzed by the aimed physiologically active substance, and the chromogenic group is liberated further by coupled exopeptidase, etc., the influence of contaminated other physiologically active substances is interferenced since the hydrolyzed substance is not measured directly
On the other hand, there is disclosed in Journal of Biochemistry 98, 1293-1299 (1985) a process wherein a peptide is used as a substrate, an amino group produced by hydrolysis is reacted with fluorescamine, and the fluorescence produced is measured. This method is good in sensitivity, but has defects in that the aimed amino group cannot be determined correctly, when other portions of the peptide are hydrolyzed by contaminated other physiologically active substances. In such a case, there is a fear of providing a large error since the measured value include the extra amino groups. Further, according to the above-mentioned reference, it is disclosed that it is possible to specify and to determine quantitatively the hydrolyzed portion by separation and determination of the reaction products using a high performance liquid chromatography. But since the absorption of peptide bond at near 220 nm is measured, there are defects in that the sensitivity is low and that many contaminants often interfere the UV-absorption.
As methods for measuring enzymes which have functions for addition or elimination of specified functional groups (e.g. phosphoric acid groups, sulfonic acid groups, sulfuric acid groups, acyl groups, etc.), there are only reported methods for using radioisotopes as disclosed, for example in Biochemistry 15, 1958-1967 (1976). According to the article of the above-mentioned Biochemistry, by using a protein such as histone, casein, or the like and adenosine 3-phosphate labelled with an isotope .sup.32 p, an enzyme which phosphorilates a protein is measured. This process is high in sensitivity, but there are defects in that it cannot be determined which portion of the protein is phosphorylated and a special devices and a careful handling are necessary due to the use of radioisotope.